Field-Flow Fractionation (FFF) was established as an orthogonal separation and characterization tool to size-exclusion chromatography (SEC) and Analytical ultracentrifugation (AUC) for proteins, polymers, nanoparticles etc. using an external, perpendicular physical field, rather than by the interaction with a stationary phase. To further determine the particles size, molar mass, etc. a MALS detector is usually configured to FFF for a better understanding of the particle properties, which are the most used FFF application research area. The robustness of FFF was demonstrated by highly consistent correlation of the HMW, LMW and monomer quantification among SEC, AUC and FFF. Here we provide an example of quantification of the mAb/protein aggregates and determination of particle sizes and solution property by FFF. Polysorbate plays critical roles in biologics formulation by stabilizing protein in solution and there are noticeable research efforts on characterization of subvisible and visible particles that are associated with degradation of the polysorbate in the biologics drug products either thorough enzymatic or oxidation reactions. The advantages of FFF were demonstrated by PS-80 and protein complex particle investigation study in which the positive charged PS-80 degradants oleic acid interacts with negative charged mAb molecules in the solution with specific pH and ionic strength. FFF can separate particles in solution based on size (diffusion vs. velocity) where the investigation particle size range are not limited to the column matrix size such as in SEC. Most importantly, the mobile phase selection is not constrained like in SEC or AUC, where the molecule solution properties can be largely influenced by using pre-determined buffer conditions. Furthermore, the loading mass can be customized according to the need and purpose of investigation in FFF, which is usually limited by column capacity in SEC and detection signal saturation in AUC. In summary, we demonstrated the utility of FFF for protein aggregation and particle characterization in protein therapeutical development with advantages over commonly used techniques such as SEC and AUC.

Field-Flow Fractionation (FFF): orthogonal SEC/AUC, particle investigation and beyond

ACS Spring 2022

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